Human germline CRISPR raises major bioethical considerations, but what about specialized issues?

Setting aside the many ethical issue in regards to the general idea of human modification itself, could this go a long way? Yes in theory it could, but there are  some very hard technological challenges that could and likely would cause problems or unacceptable outcomes at many steps along the way. These types of failures or unacceptable outcomes could easily involve actual, live people who could be harmed or die. It’ s i9000 very different than simple in vitro research so the technology side of it has been incredibly refined and robust. That’ s not going to be easy.

Human crispr challenges

What are the biggest technological roadblocks to responsible, successful (meaning safe and effective) use of CRISPR-Cas9 for heritable human genetic modification?

I see at least seven biggies.

This post is in part inspired by a section of my book on potential human use of CRISPR called GMO Sapiens .

1 ) Starting from point ACGT? Unknown initial  genomic sequence of embryos

Let’ s say we have a father-to-be, Mr. A, with a disease-causing mutation and a spouse Mrs. A who does not need that mutation. Dr . T, the leader of the scientific/medical  team, gathers normal  eggs from Mrs. A  and the sperm from Mr. A. Half of the sperm will be mutant and half will be the normal “ wildtype” so the same will be true of the embryos that result from the IVF. Dr . T’ s intention is by using CRISPR-Cas9 to revert the mutant allele back to wildtype in the embryos.

Dr . T  would  need to know the whole genome sequence of the embryos that the team will be trying to gene target. Here we have  a Catch-22 situation. Dr . T’ s team  needs to know the starting genomic sequencing of the embryos before the intervention to design the CRISPR system components including the repair template with 100% confidence (although the high level of sequence similarity between humans could enable use of the so-called “ reference” human genome for template design) and later to screen for off-target effects, but how does Dr . T  get that information without destroying the one-cell embryos? She can’ t.

So perhaps she settles for understanding Mr. and Mrs. A’ s individual genomic sequences and then infers what the embryo/fetal sequence might be like? For instance , for CRISPR guide RNA and repair template style Dr . T might assume that the mutated gene series in question will be from Mr. A in some embryos. They could also bring the reference human genome into play as well.

It definitely feels a bit like flying sightless. The best-case scenario would be that other than the veränderung in question, the parents-to-be have exactly the same sequence in the general region of interest.

2 . Mutating wildtype normal  embryos

Because the vast majority associated with possible clinical human embryo editing scenarios involve 1 parent with a heterozygous  mutation and the other without that will mutation like Mr. and Mrs. A, about half of the IVF-created embryos will have no mutation. So , Dr . Capital t cannot know if she is  attempting to edit  WT or mutant embryos prior to CRISPR-Cas9 injection. Inevitably they will be CRISPR’ ing some normal  embryos  and most likely mutating a subset  of those via Indel creation. Could it be permissible to genetically modify formerly WT human embryos that they intend to implant in a surrogate mother, potentially producing new disease-causing mutations in the worst-case scenario? I don’ t think so. I don’ t see how this might be avoided in this scenario.

Keep in mind again that when you sequence a few embryonic cells by PGD (more on that below) later during embryogenesis, a WT sequence showing up at the gene of interest could mean (A) this started out as a WT embryo or (B) this started out as a “ mutant” embryo and you successfully fixed the gene mutation. How can you tell the difference at this degree?

Let’ s say that  Dr . Capital t encodes some clever, tiny “ bar code” signpost via the CRISPR gene editing beyond the mutation modification to be an indicator during sequencing of a successful gene edit (rather than just a pre-existing WT allele). This could distinguish between correction or just starting with a WT embryo. Nevertheless , then could the indicator– say something as small as two non-codon changing basepair changes– could cause trouble? Who knows?

3. Off-target effects

CRISPR-Cas9 is great and getting better (and we even understand have an expanded toolbox with the ‘ base editor’ chemical substance modification tech), but they aren’ t perfect. There will often be a risk of it making edits in the wrong put in place addition to a chance of making wrong edits such as Indels within the right place. Since Dr . T  does not know the embryo’ t starting genomic sequences,   how does she know in the event that CRISPR-Cas9 created an off-target mutation (e. g. less than a single bp change that was undesired) or if rather that detected unexpected DNA sequence was just the exclusive sequence of the embryo to start with at some random place in the particular genome? There are a lot of naturally occurring variants including SNPs. Once again, Dr . T would need to go back to Mr. and Mrs. The. ’ s sequences and hope she can get several clarity there along with the reference human genome. However , it is possible to imagine scenarios where the team just couldn’ t make certain if a different say between a G and Capital t at one place in the billions of genomic basepairs was obviously a variant or an off-target effect.

An additional practical confounding  issue here is that Dr . T’ h team is working with human embryos with few tissues to use for screening. Ideally they need to screen for off-target effects so  they  need to be able to do whole genome sequencing (WGS). Can they accurately and reproducibly do WGS from only 1 or 2 embryonic cells (blastomeres) along with accuracy down to the single basepair resolution across the whole genome? Of course , one could wait and get more cells from the fetus later after implanting the embryo, but then in case problems arise we are talking about the possibility of an abortion entering play.

4.   PGD might usually miss mosaicism

To do the particular kinds of sequencing mentioned earlier, Dr . T is  likely to definitely do PGD and this will also be where she searches for mosaicism (i. e. some of the embryo’ s cells have got gene edits, while other cells in the same embryo do not and also there is the possibility of mosaic off-target effects). The particular team needs to know if there’ s mosaicism plus presumably if there is then they would halt the process as they usually do not want to create substantially mosaic humans who could have severe health consequences as a result.

Unfortunately if you intend to complete full genome sequencing by PGD at the 8-cell phase you are relying on just one or two cells (or perhaps a few more if you undertake PGD at the blastocyst stage) to be predictive of the entire embryo. PGD of just one or a few  cells can give you an entirely wrong view of the genotype of the other tissues in the gene edited embryo. You might well incorrectly think there is no mosaicism when in fact there is variability amongst the  cells of the embryo that you just failed to detect. Again simply by PGD you should also be looking for off-target effects and you don’ t want to limit that search only to one or one or two of  cells either. PGD is crucial, but only a part snapshot.

Another hurdle discussed in a previous blog post is coming up with a reasonable reason for using CRISPR in a clinical way in humans that will make it better than simply using already existing PGD  technology on its own (without gene editing) to screen for embryos not really possessing an inherited mutation. Why would Mrs. plus Mr. A and Dr . T even want to try gene editing instead of using PGD all by itself? It’ h difficult to come up with many scenarios where PGD alone wouldn’ t work and where gene editing would be considerably better. An excellent Nature Biotechnology piece   covers this question associated with when human modification might make sense. For instance, I thought Robin the boy wonder Lovell-Badge’ s response was very cogent. One example may be the rare situation with a parent-to-be who is homozygous for a prominent mutation or if both parents have disease-causing variations.

5. The use of thousands of human embryo for  optimization

The old saying will go that “ practice makes perfect” and that kind of emotion applies to CRISPR’ ing human embryos even if it can by no means be 100% guaranteed to be perfect. Most  attempts in human embryo editing in the lab are still likely to be useful so a knowledge base will build over time and enhance the gene editing technology and methods. It will probably get many thousands  of human embryos to optimize the machine collectively and every specific lab doing a distinct kind of gene editing may require hundreds of embryos for its own optimization.

Is it acceptable  to do such massive scale practical embryo editing simply for advancing knowledge? Also, where will you get all these eggs and embryos?   I’ mirielle a supporter of human embryonic stem cell (hESC) research and embryos remaining from IVF procedures are accustomed to make hESC (or are otherwise generally discarded), although not a tremendously huge number.   In contrast, hypothetical yearly utilization of thousands of potentially newly generated human embryos simply to enhance gene editing and/or for advancing knowledge could get ethically and practically complicated. See the recent piece on the Mitalipov lab evidently already making and using hundreds of human embryos for CRISPR.

6. Trapped in a choice of “ lesser evils”   post-implantation?  

Let’ s say somehow Dr . T successfully will get further along  and the team has got a pregnancy having a genetically modified human embryo. If a problem then comes up, what can they  do about it? The team involved in this particular work could well find themselves trapped with a dilemma as to how to deal with an adverse situation. The only options might both be challenging:   (1) continuing a risky pregnancy of an individual embryo/fetus with CRISPR-introduced  genetic errors or (2) cut it out the pregnancy. If mistakes are relatively common such clinical CRISPR research, is it OK to routinely belay such fetuses  if  problems arise?

Lastly, what if health issues  become apparent in Mr. plus Mrs. A’ s gene edited children only a lot later on down the road?

7. Unintended implications.

The genome is a complicated bush so even if you make the “ right” edit with no off-target effects, how do you know you’ ll get just the directly focused outcome you want?

Possible means to fix some problems: gene editing in germ or come cells?

The above discussion assumes the focus on gene editing conducted in one-cell embryos, however it is also in principle possible to gene edit variations in germ cells. For example , one might do CRISPR in oocytes or even primordial germ cells (assuming productive working out some of the kinks in producing such sperm plus egg-producing cells safely in humans), validate gene modification and lack of off-target effects in the cells prior to feeding, and then proceed with IVF, implantation, etc . with the gene editing now in the rear-view mirror so to speak. This could solve some of the issues mentioned earlier. At the same time this approach may have problems of its own such as the risks associated with prolonged manipulations associated with germ cells in the dish in the lab. It is also feasible that the use of cultured primordial germ cells would present unique risks as the cells change their epigenomes throughout their growth and manipulation in the lab. Still, this kind of method is another, interesting option.

Bottom line. Overall to me the big picture at this time at least any of serious technical hurdles in the way of responsible possible upcoming clinical human genetic modification. Technology will improve and we will come to see solutions to some of these problems, but it seems unlikely that every these issues can be resolved completely. Throw in the numerous thorny honest and legal issues and it seems even more difficult to imagine a future high could be responsible, safe human germline genetic modification completed with an unique, beneficial purpose. Despite all of this, I do believe that many people will go ahead and try making genetically modified individuals anyway.

Any responsible discussion of probable heritable human genetic modification needs to include dialogue upon these kinds of technical hurdles and problems. When someone is definitely aspirational about CRISPR germline use, it is worth inquiring them about these sorts of hurdles and also  about what particular positive use they had in mind  for heritable human being gene editing that transcends what embryo screening may already achieve.

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